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Drebrininhibitscofilin-inducedsevering of F-actin.

Department of Chemistry and Biochemistry, University of California, Los Angeles, California.

Abstract

Molecular cross-talk between neuronal drebrin A and cofilin is believed to be a part of the activity-dependent cytoskeleton-modulating pathway in dendritic spines. Impairments in this pathway are implicated also in synaptic dysfunction in Alzheimer's disease, Down syndrome, epilepsy, and normal aging. However, up to now the molecular interplay between cofilin and drebrin has not been elucidated. TIRF microscopy and solution experiments revealed that full length drebrin A or its actin binding core (Drb1-300) inhibits, but do not abolish cofilin-inducedsevering of actin filaments. Cosedimentation experiments showed that F-actin can be fully occupied with combination of these two proteins. The dependence of cofilin binding on fractional saturation of actin filaments with drebrin suggests direct competition between these two proteins for F-actin binding. This implies that cofilin and drebrin can either overcome or reverse the allosteric changes in F-actin induced by the competitor's binding. The ability of cofilin to displace drebrin from actin filaments is pH dependent and is facilitated at acidic pH (6.8). Pre-steady state kinetic experiments reveal that both binding and dissociation of drebrin to/from actin filaments is faster than that reported for cooperative binding of cofilin. We found, that drebrin displacement by cofilin is greatly inhibited when actin severing is abolished, which might be linked to the cooperativity of drebrin binding to actin filaments. Our results contribute to molecular understanding of the competitive interactions of drebrin and cofilin with actin filaments.

Depolymerization of yeast WT F-actin in the presence of hCof1 and Drb1-300

(A) Examples of depolymerization data used to determine the initial rates of F-actin depolymerization. Depolymerization was followed by pyrene fluorescence. As an example, part of the data used to calculate the initial rate is indicated by a solid line. Fluorescence signals of the samples after overnight (ON) incubation, corrected for instrument-related drift of the signal, are shown in the right panel. Conditions: KM2EH7.5 buffer supplemented with 1 mM DTT and 0.2 mM ATP. Final F-actin and cofilin concentrations were 2 and 1.5 μM, respectively. Concentrations of Drb1-300 in the samples are indicated next to the traces. (B) Drb1-300 decreases the extent of actin depolymerization in the presence of hCof1 compared to F-actin-cofilin control. Samples contain increasing concentrations of Drb1-300 (gel insert, left to right: 0; 0.25; 0.5; 0.75; 1; 1.5; 2 μM). Conditions and protein concentrations as in (A.) Gel analysis (SDS PAGE, 12.5%) of the samples [as shown in (A)] after overnight incubation at 4°C. Supernatants and pellets were separated by high speed centrifugation and a representative gel of supernatant samples stained with Coomassie Blue is shown. Protein bands are marked as yA–yeast actin; D1–300–Drb1-300; hC1–human cofilin 1. Amounts of actin in supernatants were quantified using yeast actin standards (labeled as S0.5; S1; S2, numbers (0.5, 1, and 2) correspond to the actin concentration in μM). Dependence of yeast WT F-actin (2 μM) depolymerization rates in the presence of human cofilin-1 (1.5 μM) on the concentration of Drb1-300 construct (average of two independent experiments). Conditions: 1× KM2EH7.5 buffer supplemented with 0.2 mM ATP and 1mM DTT.

(A) Dependence of kobs on the concentration of free Drb1-300-DABMI. Inset: Time course of a decrease in FRET signal upon interaction of skeletal F-actin-IAEDANS (2 μM) with 5.5 μM of Drb1-300-DABMI (raw data). (B) Increase in a FRET signal upon Drb1-300-DABMI (1.5 μM) dissociation from IAEDANS-labeled actin filaments due to its competition with unlabeled Drb1-300 (15 μM) (raw data).

Different scenarios of drebrin-cofilin competition for F-actin binding

In red—F-actin sites unavailable for cofilin binding either due to their protection by drebrin or due to the propagation of 40 nm change in helical repeat [; ] from a drebrin cluster; green—F-actin sites available for cofilin binding; yellow circles—cofilin; dark blue line—Drb1-300 (interacts with 3 actin protomers). (A) Direct and allosteric inhibition of cofilin binding by drebrin. Undecorated actin region next to the drebrin cluster is unavailable for cofilin binding due to the propagation of morphological changes in actin filaments. Such allosteric effect, together with direct competition for F-actin binding, would lead to a dramatic decrease in cofilin’s affinity to drebrin-decorated filaments (not observed); (B) Direct competition. Drebrin-decorated actin region is protected from cofilin binding due to their competition for binding sites on F-actin. Cofilin affinity to F-actin is only mildly reduced in the presence of saturating concentrations of drebrin allowing it to bind to actin sites adjacent to drebrin clusters (consistent with our data); (C) Direct competition with the possibility of cofilin binding to F-actin within drebrin clusters. Total combined occupancy of drebrin and cofilin >1 suggests the possibility of drebrin and cofilin co-binding to F-actin, leading to a ternary complex formation (suggested by our data).